Article pubs.acs.org/JAFC
Stereospecific Analysis of Triacylglycerol and Phospholipid Fractions of Five Wild Freshwater Fish from Poyang Lake Lin Lei,† Jing Li,† Guang-Yan Li, Jiang-Ning Hu, Liang Tang, Rong Liu, Ya-Wei Fan, and Ze-Yuan Deng* State Key Lab of Food Science and Technology, Institute for Advanced Study, Nanchang University, Nanchang, Jiangxi, 330047, China ABSTRACT: The fatty acids (FA) compositions and positional distributions in triacylglycerols (TAG) and phospholipids (PL) of five wild freshwater fish (Squaliobarbus curriculus, Erythroculter ilishaeformis, Pseudobagrus f ulvidraco, Bostrichthys sinensis, and Siniperca kneri Garman) from Poyang Lake (the largest freshwater lake of China) were studied. For TAG, S. kneri German had the highest content (13.59%) of n − 3 polyunsaturated fatty acids (PUFA) and E. ilishaeformis had the lowest ratio of (n − 6)/(n − 3) (0.65). PL had a high content of PUFA, which declined in the order of phosphatidylethanolamine (PE) > phosphatidylcholine (PC) > TAG. 9c11t-18:2 accounted for 6.38−50.77% of total conjugated linoleic acids (CLA). The highest level of odd-branched chain fatty acids (OBCFA) was 26.7% in B. sinensis. The study revealed that the distribution of FA among the sn positions was not random: monounsaturated fatty acids (MUFA) and PUFA preferred positions 1 and 3 and saturated fatty acids (SFA) position 2 of TAG, while SFA and MUFA predominated over sn-1-PL and PUFA over sn-2-PL. KEYWORDS: Poyang Lake, wild freshwater fish, triacylglycerol, phosphatidylcholine, phosphatidylethanolamine, stereospecific analysis
■
INTRODUCTION Fish lipids have been under scientific research as a good source of n − 3 and n − 6 polyunsaturated fatty acids (PUFA), which have effects on retinal and neural development for infants, inflammation, and immunity processes as well as prevention of cardiac and circulatory disorders.1−3 Although freshwater fish in general contain higher proportions of n − 6 PUFA (2.42− 21.92%) than marine species (0.43−14.2%), they also have excellent levels of n − 3 PUFA, such as 20:5n-3 (1.15−13.8%)) and 22:6n-3 (0.94−24.8%), with a ratio of (n − 6)/(n − 3) PUFA varing between 0.04 and 4.55.4,5 Fish PUFA may vary by differences in location, environment, season, gender, diet, and species. In fact, Jabeen and Chaudhry related the high amount of PUFA in freshwater fish with their feed preference to phytoplanktons, which are usually rich in essential fatty acids (FA), 18:2n-6 and 18:3n-3.6,7 Compared with marine species, freshwater fish are more capable of desaturating and elongating 18:2n-6 and 18:3n-3 into long-chain PUFA (LCPUFA, ≥C20) due to their higher activity of Δ6D (FA desaturase) gene expression.8,9 Poyang Lake, as a main water source of the Yangtze River, is the largest freshwater lake in China and harbors about 133 species of fish. Thus, given the dietary and geographic effects on fish FA, the fish PUFA in Poyang Lake is distinguished from other regions studied before. About 30 000 tons of fish in Poyang Lake have been caught annually in the 21st century and are commercially important for food in the Jiangxi province. However, little attention has been paid to the fish FA compositions of Poyang Lake. Currently, PUFA like 20:5n-3 and 22:6n-3 from deep-sea fish are developed as nutraceuticals, which are relatively expensive due to the great depth of the deep sea, making it hard to exploit. Since freshwater fish contain a good amount of n − 3 and n − 6 PUFA, how to adequately utilize freshwater fish lipids from Poyang Lake is highly important and promising. © 2012 American Chemical Society
On the other hand, the bioavailability of FA is affected by the structures of dietary triacylglycerols (TAG) and phospholipids (PL). Due to the specific hydrolysis of pancreatic lipase on TAG and phospholipase A2 on PL, respectively, dietary lipids are mainly absorbed as sn-2-monoacylglycerols (sn-2-MAG), sn-1-lysophospholipids, and free FA split from the 1 and 3 positions of TAG or 2 position of PL, but absorption in the body gets more difficult for released long-chain saturated fatty acids (≥C14) because they tend to form insoluble soaps with magnesium and calcium.10 The hydrolyzed products (≥C14) are absorbed, subjected to re-esterification, and then incorporated into the chylomicrometers as newly formed TAG and PL in the enterocytes. Therefore, FA of the sn-2-position and sn-1position from dietary TAG and PL, respectively, are retained in plasma TAG, whereas FA split off may be partially replaced by endogenous FA.10,11 The compositions, distributions, carbon chain length, and degree of unsaturation of FA vary significantly from species to species in fish lipids, which generally affect the nutritional, digestive, absorptive, and metabolic properties of dietary lipids.12 Thus, to shed more light on the nutritional and biological effects of fish lipids, it is of significance to gain deeper understanding of structural FA distributions. We have early shown that wild freshwater fish, namely, Squaliobarbus curriculus, Erythroculter ilishaeformis, Pseudobagrus f ulvidraco, Bostrichthys sinensis, and Siniperca kneri Garman of Poyang Lake have higher total PUFA than freshwater species from other regions, and this value is similar to seawater fish.13,14 For comprehensive details of the fish lipids as mentioned above, the aim of this study was to determine the FA profiles and stereospecific analysis of TAG, phosphatidylcholine (PC), Received: Revised: Accepted: Published: 1857
November 9, 2011 January 13, 2012 January 13, 2012 January 14, 2012 dx.doi.org/10.1021/jf204584t | J. Agric.Food Chem. 2012, 60, 1857−1864
Journal of Agricultural and Food Chemistry
Article
Table 1. Total Lipid (% Wet Weight) and Lipid Classes (% Total Lipid) from Five Wild Freshwater Fish in Poyang Lakea TL S. curriculus E. ilishaeformis P. f ulvidraco B. sinensis S. kneri Garman
2.10 1.73 1.29 0.51 0.37
± ± ± ± ±
0.19d 0.20c 0.22b 0.17a 0.08a
TAG 80.86 65.69 79.59 59.48 55.64
± ± ± ± ±
PC
0.43e 0.39d 0.50c 0.36b 0.44a
3.75 4.06 7.79 11.16 11.81
± ± ± ± ±
PE 0.33a 0.26a 0.19b 0.30c 0.45d
2.04 2.80 4.70 5.07 5.49
± ± ± ± ±
0.19a 0.21b 0.23c 0.32c 0.18d
Mean values ± SD (n = 3). Values in the same column with different letters show significant differences (p < 0.05). TL, total lipids; TAG, triacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine.
a
by Ichihara et al.,18 and another aliquot of purified TAG was methylated according to Cruz-Hernandez et al.19 The mean composition of each FA in positions 1 and 3 can be calculated from its concentrations in the intact TAG and sn-2-MAG according to the following relationship
and phosphatidylethanolamine (PE) of these five wild freshwater fish from Poyang Lake.
■
MATERIALS AND METHODS
Materials and Chemicals. Five wild freshwater fish, S. curriculus, E. ilishaeformis, P. f ulvidraco, B. sinensis, and S. kneri Garman, were captured in May within Poyang Lake of Jiangxi province, China. Six individuals of each species were transported in ice to the laboratory. After removal of head, skin, viscera, and bone they were homogenized by a blender and then stored at −80 °C until analyzed. Fish lipids were extracted by a previous method.15 PL standards from egg yolk including PC (purity > 99%), PE (purity > 98%), lysophosphatidylcholine (LPC, purity > 99%), and lysophosphatidylethanolamine (LPE, purity > 99%), lipase from porcine pancreas (Type II), phospholipase A2 from porcine pancreas, and (trimethylsilyl) diazomethane solution were from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Standards of TAG, diacylglycerols, monoacylglycerols, and free FA were collected using modified Hita method from fish lipids.16 Standard fatty acid methyl esters (FAME, #463) spiked with a mixture of four positional CLA isomers (#UC-59 M) were obtained from Nu-Chek Prep Inc. (Elysian, MN). Silica gel (ZCX-II, 54−74 μm) and silica gel GF254 TLC plates (20 × 20 cm) were obtained from Haiyang Chemical Group (Qingdao, China). n-Hexane used in GC was purchased from Merck (Darmstadt, Germany), and other solvents were analytical reagent grade. Separation of Lipids by Adsorption Chromatography. Silica gel containing 5% water was prepared beforehand. A glass column containing silica gel was conditioned using 3 column volumes of petroleum ether (bp 40−60 °C)/ether (87:13, v/v), and then the fish lipids were applied onto the column according to the published method with modifications.17 The neutral lipids tended to elute first with the solvents mentioned above (87:13, v/v) by 2 column volumes, followed by glycolipids with 2 column volumes of acetone and finally PL (methanol, 8 column volumes). Butylated hydroxytoluene (0.02%, BHT) was added to the solvents to prevent oxidation of PUFA. In each fraction about 1/3 of the column volume was collected into a tube, evaporated under vacuum (40 °C), and determined by TLC using n-hexane/ether/acetic acid (75:25:1, v/v/v) as developing solvent for neutral lipids while chloroform/methanol/water (65:25:4, v/v/v) was used for PL. The plates were allowed to dry under N2 atmosphere and visualized by dichlorofluorescein. Lipids fractions were identified by comparison with TAG, PC, and PE standards. Fractions containing homogeneous TAG, PC, and PE were accumulated. Stereospecific Analysis of TAG. The method of Hita et al. with modification was employed to determine FA in position 2 of TAG: an aliquot (10 mg) of purified TAG, 10 mL of buffer solution tris-HCl (1M, pH 7.6), and 2.5 mL of 0.05% bile salts solution. One milliliter of 2.2% calcium chloride solution and 10 mg of pancreatic lipase were placed in a centrifuge tube and shaken vigorously for 1 min. Later, the mixture was incubated for 3 min at 37 °C and shaken intensively for 30 s, and then this procedure was repeated another two times. Afterward, 6 mL of diethyl ether was added to stop the reaction and the mixture was centrifuged at 4200 rpm for 5 min. The upper layer was dried under N2 atmosphere and analyzed by TLC and GC.16 sn-2-MAG obtained was subjected to methylation described
positions 1 and 3 mol % = (TAG mol % × 3 − sn‐2‐MAG mol %)/2 Stereospecific Analysis of PC and PE. The method of Robertson and Lands with little modification was employed to determine FA in positions 1 and 2 of PC (PE): an aliquot (5 mg) of purified PC (PE), 0.1 mL of phospholipase A2 solution, and 2 mL of diethyl ether were placed in a centrifuge tube; then it was shaken vigorously for 3 h to be hydrolyzed thoroughly with 0.02% BHT under N2 atmosphere at 25 °C. The mixture was analyzed by TLC and GC.20 sn-1-LPC (sn-1-LPE) and intact PC (PE) were subjected to transesterification as described by Cruz-Hernandez et al.,19 and free FA (in position 2) released from PC (PE) were methylated by the previous method.21 GC Analysis. The FAME were analyzed by a GC equipped with a flame ionization detector and a fused silica capillary column (100 m × 0.25 mm × 0.2 μm) coated with 100% cyanopropyl polysiloxane (CPSil 88, Chrompack; Middelburg, The Netherlands). The temperature program was 86 min in total: the initial temperature of the oven was 45 °C for 4 min, increased to 175 °C at a rate of 13 °C/min, maintained for 27 min, further raised to 215 °C at a rate of 4 °C/min, and finally kept at this temperature for 35 min.19 Analysis of all peaks was accomplished by comparison of their retention time with FAME standards. All samples were carried out in triplicate. Statistical Analysis. Fatty acid compositions were analyzed with one-way analysis of variance, and mean values were compared using Duncan’s test. The significance level was set at p < 0.05. All statistical analyses were carried out using SPSS 13.0 software for Windows.
■
RESULTS Lipid Contents. The lipid contents (%) of five wild freshwater fish are presented in Table 1. The results showed the total lipid contents differed among different species, and TAG composed the major part of the total lipids. The maximal values of total lipids (2.10%) and TAG (80.86%) were determined in S. curriculus, with a minimum in PC (3.75%) and PE (2.04%), whereas S. kneri Garman was lowest in total lipids (0.37%) and TAG contents (55.64%) but highest in PC (11.81%) and PE (5.49%). In general, an increase in the total lipids level was accompanied with an increase in TAG but a decrease in PL contents. Total FA Compositions of TAG and PL in Five Wild Freshwater Fish. Table 2 illustrates the total FA profile in TAG, PC, and PE from S. curriculus, E. ilishaeformis, P. f ulvidraco, B. sinensis, and S. kneri Garman. Generally, significant differences were observed for the majority of the FA among TAG, PC, and PE. In each species total saturated fatty acids (SFA) and total monounsaturated fatty acids (MUFA) mainly increased in the order PE < PC < TAG, whereas total PUFA declined as 1858
dx.doi.org/10.1021/jf204584t | J. Agric.Food Chem. 2012, 60, 1857−1864
0.15 ± 0.02a
0.15 ± 0.00b
0.06 ± 0.00b
0.25 ± 0.00b
0.08 ± 0.00a
2.66 ± 0.04b
0.29 ± 0.00b
0.13 ± 0.00a
0.06 ± 0.00
0.21 ± 0.00c
0.05 ± 0.01a
0.49 ± 0.03c
0.14 ± 0.02b
9.65 ± 0.06c
0.35 ± 0.04b
0.11 ± 0.01a
t16:1n-8
t16:1n-7
16:1n-9
16:1n-8
16:1n-7
1859
16:1n-6
17:1(a)
0.05 ± 0.00a
12.53 ± 0.57b
2.58 ± 0.02b
0.14 ± 0.03ab
0.13 ± 0.03ab
0.18 ± 0.00a
26.94 ± 0.10c
4.08 ± 0.02c
0.26 ± 0.03c
0.19 ± 0.02b
0.24 ± 0.04
0.51 ± 0.03b
18:1n-9
18:1n-7
18:1n-6
18:1n-5
19:1
20:1n-15
20:1n-12
20:1n-9
22:1n-9
19.47 ± 0.48b
18:1n-12
0.08 ± 0.00a
0.06 ± 0.00a
0.20 ± 0.01c
t18:1n-7
0.12 ± 0.07a
0.19 ± 0.01ab
0.27 ± 0.05b
t18:1n-9
43.90 ± 0.14c
0.18 ± 0.00a
0.42 ± 0.02b
0.73 ± 0.04c
17:1n-8
24:1n-9
0.22 ± 0.01b
0.10 ± 0.02a
0.18 ± 0.02b
17:1n-10
total cis MUFA
0.03 ± 0.00a
0.20 ± 0.01b
17:1n-12
12.45 ± 0.28a
0.18 ± 0.00a
0.06 ± 0.03a
0.17 ± 0.03b
2.38 ± 0.03a
7.66 ± 0.19a
0.10 ± 0.01b
0.15 ± 0.01a
0.04 ± 0.00a
0.05 ± 0.00a
17:1(b)
0.12 ± 0.03a
0.06 ± 0.00a
1.33 ± 0.02a
0.07 ± 0.00a
29.50 ± 0.00a
0.31 ± 0.00b
0.17 ± 0.02b
14:1n-5
0.05 ± 0.00a
0.26 ± 0.00c
20:0
0.51 ± 0.00b
29.72 ± 0.39a
0.12 ± 0.01a
0.17 ± 0.05a
19:0
16.62 ± 0.26b
31.59 ± 0.52b
4.41 ± 0.02a
4.65 ± 0.08a
18:0
total SFA
0.02 ± 0.00a
0.08 ± 0.00b
iso 18:0
1.29 ± 0.03c
0.71 ± 0.00a
0.87 ± 0.05b
17:0
0.24 ± 0.03
anti 17:0
0.62 ± 0.02a
24:0
0.74 ± 0.01b
0.97 ± 0.02c
iso 17:0
9.61 ± 0.23a
22.36 ± 0.34c
19.68 ± 0.24b
16:0
0.05 ± 0.00a
0.26 ± 0.00a
0.10 ± 0.00b
0.22 ± 0.00c
iso 16:0
0.13 ± 0.00a
0.07 ± 0.01
0.43 ± 0.01b
0.79 ± 0.01c
15:0
0.36 ± 0.00c
0.12 ± 0.00
anti 15:0
22:0
0.07 ± 0.00a
0.42 ± 0.01b
iso 15:0
0.19 ± 0.01a
PE
23:0
0.45 ± 0.00b
0.08 ± 0.00
2.36 ± 0.04c
14:0
13:0
iso 14:0
0.02 ± 0.00
0.08 ± 0.00
iso 13:0
PC
S. curriculus
0.15 ± 0.00
TAG
12:0
FA
39.99 ± 0.78c
0.07 ± 0.01
0.51 ± 0.01b
0.10 ± 0.00
0.11 ± 0.00
0.12 ± 0.05a
3.60 ± 0.09c
25.91 ± 0.52c
0.08 ± 0.01b
0.21 ± 0.02b
1.01 ± 0.02b
0.07 ± 0.00a
0.11 ± 0.01
0.08 ± 0.01a
0.17 ± 0.05a
0.27 ± 0.01c
7.61 ± 0.12c
0.43 ± 0.01c
0.20 ± 0.02b
0.06 ± 0.00
34.79 ± 0.72b
0.03 ± 0.00
0.80 ± 0.03b
0.13 ± 0.00b
0.32 ± 0.00c
0.30 ± 0.00b
6.02 ± 0.11a
0.07 ± 0.03a
1.05 ± 0.03b
0.97 ± 0.03c
21.04 ± 0.52b
0.19 ± 0.05b
0.82 ± 0.02c
0.09 ± 0.02b
0.43 ± 0.00c
2.23 ± 0.05c
0.05 ± 0.01
0.06 ± 0.00b
0.02 ± 0.01
0.09 ± 0.00b
TAG
21.51 ± 0.05b
0.24 ± 0.02a
0.30 ± 0.04b
2.58 ± 0.04b
14.65 ± 0.29b
0.03 ± 0.01a
0.19 ± 0.04ab
0.73 ± 0.29ab
0.05 ± 0.02a
0.22 ± 0.02a
0.20 ± 0.01b
2.62 ± 0.01b
0.20 ± 0.01b
0.79 ± 0.02b
0.29 ± 0.02c
33.71 ± 0.18ab
0.36 ± 0.03a
0.05 ± 0.01a
0.09 ± 0.01a
0.16 ± 0.01a
5.53 ± 0.14a
0.04 ± 0.00a
0.82 ± 0.02a
0.72 ± 0.01b
24.41 ± 0.03c
0.12 ± 0.01ab
0.57 ± 0.01b
0.01 ± 0.01a
0.11 ± 0.01b
0.71 ± 0.01b
0.02 ± 0.01a
0.02 ± 0.02a
PC
E. ilishaeformis
11.86 ± 0.15a
0.25 ± 0.02a
0.12 ± 0.01
0.14 ± 0.00a
2.07 ± 0.00a
7.41 ± 0.12a
0.15 ± 0.02
0.07 ± 0.00b
0.13 ± 0.00a
0.24 ± 0.00a
0.12 ± 0.00b
0.26 ± 0.01a
0.06 ± 0.01a
1.21 ± 0.01a
0.09 ± 0.01a
0.60 ± 0.01a
0.07 ± 0.01a
32.40 ± 0.13a
0.30 ± 0.03a
0.19 ± 0.02b
0.50 ± 0.01c
18.69 ± 0.33b
1.47 ± 0.02c
0.61 ± 0.02a
10.21 ± 0.19a
0.05 ± 0.00a
0.14 ± 0.01a
0.05 ± 0.01a
0.20 ± 0.00a
PE
42.41 ± 0.86c
0.05 ± 0.00
1.20 ± 0.01c
1.26 ± 0.01b
0.10 ± 0.02
0.17 ± 0.01
0.05 ± 0.00a
0.25 ± 0.01b
3.90 ± 0.09c
24.83 ± 0.58c
0.07 ± 0.00a
0.10 ± 0.01b
0.33 ± 0.03b
0.99 ± 0.03c
0.10 ± 0.01b
0.22 ± 0.01b
0.12 ± 0.01b
0.23 ± 0.01b
0.29 ± 0.03b
8.26 ± 0.20c
0.66 ± 0.03b
0.17 ± 0.03b
0.19 ± 0.00
36.19 ± 0.78c
0.13 ± 0.00
0.56 ± 0.00b
0.09 ± 0.00b
0.27 ± 0.00c
0.43 ± 0.06b
5.85 ± 0.11b
0.04 ± 0.01a
1.23 ± 0.03c
1.73 ± 0.03c
21.10 ± 0.52b
0.41 ± 0.01c
1.13 ± 0.02c
0.17 ± 0.01b
0.45 ± 0.07b
2.30 ± 0.06c
0.06 ± 0.00
0.09 ± 0.00b
0.02 ± 0.00
0.14 ± 0.00b
TAG
19.84 ± 0.01b
0.46 ± 0.01a
0.19 ± 0.01a
0.08 ± 0.02a
0.08 ± 0.02a
3.04 ± 0.02b
10.94 ± 0.04b
0.07 ± 0.00a
0.05 ± 0.00a
0.33 ± 0.00b
0.52 ± 0.01b
0.03 ± 0.00a
0.09 ± 0.01a
0.09 ± 0.01a
0.23 ± 0.01b
0.32 ± 0.07b
3.27 ± 0.00b
0.74 ± 0.01c
0.32 ± 0.00c
30.89 ± 0.68b
0.32 ± 0.03a
0.03 ± 0.04a
0.07 ± 0.01a
0.23 ± 0.00a
4.76 ± 0.03a
0.02 ± 0.01a
0.73 ± 0.01a
0.97 ± 0.01b
21.52 ± 0.72b
0.18 ± 0.02b
0.95 ± 0.02b
0.02 ± 0.02a
0.12 ± 0.02a
0.92 ± 0.02b
0.02 ± 0.01a
0.02 ± 0.01a
PC
P. f ulvidraco
14.70 ± 0.58a
0.60 ± 0.04b
0.13 ± 0.03a
0.16 ± 0.03a
2.26 ± 0.01a
9.54 ± 0.47a
0.13 ± 0.01b
0.10 ± 0.01b
0.23 ± 0.00a
0.30 ± 0.00a
0.18 ± 0.01a
0.05 ± 0.01a
1.34 ± 0.00a
0.18 ± 0.01a
0.05 ± 0.01a
27.12 ± 0.17a
0.32 ± 0.05a
0.17 ± 0.01b
0.74 ± 0.07c
16.84 ± 0.20c
0.19 ± 0.01b
1.01 ± 0.01b
0.72 ± 0.03a
6.33 ± 0.00a
0.07 ± 0.0a
0.16 ± 0.00a
0.55 ± 0.00a
PE
31.33 ± 0.78c
0.07 ± 0.02a
0.49 ± 0.02b
0.25 ± 0.01
0.13 ± 0.01a
3.25 ± 0.09b
14.22 ± 0.45c
0.76 ± 0.00c
1.20 ± 0.04c
0.46 ± 0.03b
0.37 ± 0.04b
0.56 ± 0.07b
10.41 ± 0.22c
0.34 ± 0.02ab
0.18 ± 0.02
40.07 ± 0.97c
0.46 ± 0.08a
0.10 ± 0.01a
0.31 ± 0.01a
3.61 ± 0.07a
1.57 ± 0.05ab
1.85 ± 0.04c
18.91 ± 0.66b
0.52 ± 0.01c
4.55 ± 0.13c
0.22 ± 0.02b
1.25 ± 0.04c
4.59 ± 0.15c
0.22 ± 0.01b
1.46 ± 0.05b
0.04 ± 0.02
0.41 ± 0.01b
TAG
Table 2. Total Fatty Acids Compositions (%) in TAG, PC, and PE of Five Wild Freshwater Fish from Poyang Lakea
15.61 ± 0.19b
0.23 ± 0.05b
0.15 ± 0.06a
0.10 ± 0.03a
3.16 ± 0.08b
8.15 ± 0.28b
0.31 ± 0.06a
0.68 ± 0.02b
0.02 ± 0.01a
0.10 ± 0.02a
0.37 ± 0.02a
2.66 ± 0.02b
0.38 ± 0.02b
33.98 ± 0.06b
0.23 ± 0.02a
0.14 ± 0.03a
0.38 ± 0.01b
5.74 ± 0.15b
1.88 ± 0.18b
1.05 ± 0.01b
20.65 ± 0.11c
0.26 ± 0.03b
2.40 ± 0.08b
0.03 ± 0.01a
0.22 ± 0.03b
0.92 ± 0.04b
0.01 ± 0.01a
0.07 ± 0.00a
0.02 ± 0.01a
PC
B. sinensis
10.89 ± 0.15a
0.46 ± 0.03c
0.13 ± 0.04a
0.15 ± 0.02a
2.63 ± 0.02a
5.36 ± 0.12a
0.08 ± 0.04
0.09 ± 0.03
0.15 ± 0.05b
0.52 ± 0.05a
0.37 ± 0.04a
1.21 ± 0.11a
0.15 ± 0.11a
27.97 ± 0.39a
0.32 ± 0.11a
0.25 ± 0.11a
1.12 ± 0.01c
14.57 ± 0.47c
1.37 ± 0.04a
0.75 ± 0.02a
8.82 ± 0.26a
0.02 ± 0.02a
0.44 ± 0.01a
0.09 ± 0.02a
0.22 ± 0.01a
PE
29.34 ± 0.86c
0.07 ± 0.01
0.18 ± 0.04
0.55 ± 0.03b
0.08 ± 0.04
0.13 ± 0.04
0.25 ± 0.04b
0.22 ± 0.04b
5.38 ± 0.20c
11.62 ± 0.44c
0.17 ± 0.01b
0.22 ± 0.00c
0.36 ± 0.01c
0.86 ± 0.02b
0.20 ± 0.01
0.47 ± 0.01
0.14 ± 0.02
0.33 ± 0.09a
0.52 ± 0.01c
7.99 ± 0.08c
0.18 ± 0.06b
0.54 ± 0.07c
0.25 ± 0.04b
0.07 ± 0.00
35.18 ± 0.86a
0.17 ± 0.03b
0.37 ± 0.03b
0.13 ± 0.03
0.24 ± 0.01a
0.78 ± 0.02b
4.93 ± 0.12b
0.12 ± 0.01b
1.83 ± 0.02a
0.17 ± 0.02b
2.23 ± 0.02c
16.29 ± 0.05a
1.10 ± 0.02c
1.58 ± 0.04c
0.61 ± 0.05b
1.41 ± 0.05c
2.60 ± 0.07b
0.21 ± 0.01b
0.18 ± 0.00b
0.04 ± 0.00
0.18 ± 0.01
TAG
PC
14.13 ± 0.08b
0.80 ± 0.00c
0.07 ± 0.01a
0.07 ± 0.00a
2.49 ± 0.14a
8.30 ± 0.10b
0.08 ± 0.02a
0.03 ± 0.01a
0.28 ± 0.00b
0.45 ± 0.02a
0.31 ± 0.04a
0.43 ± 0.01b
1.97 ± 0.00b
0.06 ± 0.00ab
0.11 ± 0.00a
0.11 ± 0.00
0.25 ± 0.01b
34.97 ± 0.09a
0.11 ± 0.03b
0.15 ± 0.04a
0.26 ± 0.02a
3.74 ± 0.21a
0.03 ± 0.00a
1.77 ± 0.14a
0.03 ± 0.01a
0.80 ± 0.14a
26.00 ± 0.42b
0.27 ± 0.03b
0.88 ± 0.14b
0.05 ± 0.02a
0.22 ± 0.02b
0.62 ± 0.07a
0.02 ± 0.01a
0.01 ± 0.01a
S. kneri Garman PE
10.37 ± 0.36a
0.19 ± 0.01a
0.11 ± 0.01a
0.16 ± 0.04ab
3.29 ± 0.27b
4.37 ± 0.03a
0.12 ± 0.01a
0.12 ± 0.01b
0.20 ± 0.01a
0.25 ± 0.06a
1.19 ± 0.07b
0.27 ± 0.02a
1.20 ± 0.04a
0.03 ± 0.01a
0.41 ± 0.02b
0.07 ± 0.01a
37.46 ± 0.18b
0.02 ± 0.01a
0.11 ± 0.01a
0.25 ± 0.06a
1.65 ± 0.01c
17.78 ± 0.30c
2.64 ± 0.01c
1.32 ± 0.08b
12.52 ± 0.08a
0.10 ± 0.02a
0.52 ± 0.03a
0.12 ± 0.01a
0.46 ± 0.01a
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf204584t | J. Agric.Food Chem. 2012, 60, 1857−1864
1860
0.08 ± 0.01a
0.10 ± 0.00a
0.36 ± 0.05b
total trans DUFA
0.08 ± 0.00a
0.09 ± 0.00a
5.04 ± 0.08b
0.07 ± 0.00a
0.37 ± 0.00a
0.71 ± 0.01b
15.27 ± 0.16b
0.77 ± 0.01b
2.20 ± 0.09b
1.89 ± 0.00b
0.23 ± 0.01a
7.54 ± 0.12c
1.26 ± 0.05b
12.18 ± 0.30b
0.03 ± 0.01
0.29 ± 0.02
0.48 ± 0.04b
8.28 ± 0.04c
0.21 ± 0.01b
0.40 ± 0.00b
0.33 ± 0.01a
2.48 ± 0.10a
0.23 ± 0.00a
0.53 ± 0.05a
4.27 ± 0.05c
0.34 ± 0.00b
2.10 ± 0.08a
0.63 ± 0.00a
2.24 ± 0.20a
11t13t18:2
8t10t/ 9t11t/ 10t12t18:2
total CLA
18:2n-6
18:3n-6
20:2n-6
20:3n-6
20:4n-6
22:2n-6
22:4n-6
22:5n-6
18:3n-3
20:3n-3
20:5n-3
22:3n-3
22:5n-3
22:6n-3
1.06 ± 0.01b
1.60 ± 0.04b
3.16 ± 0.01a
12.45 ± 0.21a
9.59 ± 0.33a
1.30 ± 0.64c
0.70 ± 1.05a
5.94 ± 0.19b
total n − 6 PUFA
total n − 3 PUFA
(n − 6)/ (n − 3) PUFA
total ((n −6) + (n − 3))/ SFA total OBCFA
23.10 ± 0.38b
24.44 ± 0.19b
23.03 ± 0.62a
total PUFA
47.81 ± 0.57b
11c13t18:2
3.42 ± 0.08a
1.97 ± 0.10c
0.83 ± 0.06a
28.81 ± 2.71c
25.36 ± 0.33c
55.25 ± 3.06c
22.93 ± 0.28b
2.33 ± 0.12c
3.15 ± 0.05b
0.40 ± 2.54c
1.09 ± 0.06a
4.87 ± 0.15c
1.26 ± 0.07c
16.06 ± 0.03c
0.76 ± 0.02b
0.46 ± 0.01c
2.95 ± 0.21a
0.09 ± 0.00a
0.16 ± 0.01b
9c11t-18:2
6.29 ± 0.10b
0.64 ± 0.00a
0.65 ± 0.00a
13.52 ± 0.24a
8.85 ± 0.10a
22.89 ± 0.35a
6.01 ± 0.13a
1.55 ± 0.01a
0.01 ± 0.00
3.26 ± 0.05a
0.39 ± 0.01b
2.30 ± 0.04c
1.42 ± 0.03a
0.56 ± 0.00a
3.62 ± 0.00a
0.50 ± 0.01b
0.31 ± 0.01c
0.10 ± 0.00b
2.33 ± 0.05c
0.26 ± 0.03b
0.22 ± 0.02b
0.01 ± 0.02
0.03 ± 0.04
0.26 ± 0.02b
0.07 ± 0.02
0.09 ± 0.02
0.10 ± 0.02b
0.49 ± 0.01a
TAG
3.91 ± 0.29a
1.26 ± 0.02b
0.76 ± 0.00b
24.16 ± 0.49b
18.29 ± 0.41b
42.58 ± 0.90b
15.44 ± 0.36b
1.54 ± 0.01a
6.12 ± 0.13b
0.21 ± 0.11a
0.86 ± 0.01b
4.23 ± 0.18b
1.01 ± 0.01b
0.06 ± 0.01b
10.69 ± 0.24b
0.53 ± 0.00b
0.23 ± 0.00b
0.05 ± 0.00a
1.49 ± 0.01b
3.73 ± 0.01a
1.66 ± 0.01c
0.80 ± 0.00c
29.88 ± 0.40c
23.87 ± 0.21c
53.84 ± 0.57c
23.85 ± 0.31c
2.29 ± 0.06b
3.03 ± 0.05a
0.22 ± 0.00a
0.48 ± 0.01a
6.24 ± 0.21c
1.93 ± 0.04c
0.03 ± 0.00a
14.16 ± 0.07c
0.41 ± 0.01a
0.21 ± 0.00a
0.89 ± 0.01a
0.09 ± 0.04a
0.09 ± 0.04a
0.12 ± 0.01a
0.12 ± 0.01a
0.87 ± 0.00b
PE
0.01 ± 0.01a
0.01 ± 0.01a
1.29 ± 0.01c
PC
E. ilishaeformis
8.14 ± 0.03c
0.48 ± 0.00a
1.00 ± 0.01b
8.77 ± 0.08a
8.78 ± 0.13a
18.38 ± 0.33a
3.17 ± 0.04a
1.31 ± 0.00a
1.80 ± 0.02a
0.34 ± 0.04a
2.15 ± 0.05c
0.91 ± 0.01a
0.70 ± 0.00a
0.04 ± 0.00a
2.73 ± 0.05a
0.33 ± 0.01a
0.73 ± 0.01b
0.13 ± 0.00b
3.20 ± 0.06c
0.50 ± 0.10b
0.38 ± 0.04b
0.05 ± 0.03
0.07 ± 0.03a
0.34 ± 0.01b
0.13 ± 0.02b
0.12 ± 0.01b
0.08 ± 0.00
0.61 ± 0.01b
TAG
4.53 ± 0.01b
1.32 ± 0.01b
1.10 ± 0.01c
19.36 ± 0.32b
21.27 ± 0.24b
40.94 ± 0.63b
11.90 ± 0.17b
1.80 ± 0.01b
4.58 ± 0.10b
0.31 ± 0.01a
0.77 ± 0.01b
2.25 ± 0.01b
1.59 ± 0.02b
0.05 ± 0.03a
13.84 ± 0.20b
0.60 ± 0.01b
0.80 ± 0.01c
0.05 ± 0.00a
2.10 ± 0.03b
0.19 ± 0.05a
0.16 ± 0.00a
0.03 ± 0.05a
0.11 ± 0.02a
0.05 ± 0.00a
0.06 ± 0.02a
0.70 ± 0.00c
PC
P. f ulvidraco
3.70 ± 0.02a
2.03 ± 0.01c
0.68 ± 0.04a
32.87 ± 0.26c
22.22 ± 1.01b
55.23 ± 0.74c
28.26 ± 0.22c
2.01 ± 0.01c
1.84 ± 0.04a
0.32 ± 0.01a
0.44 ± 0.07a
2.46 ± 0.03c
1.88 ± 0.03c
15.44 ± 0.90b
0.59 ± 0.00b
0.62 ± 0.02a
1.23 ± 0.03a
0.14 ± 0.00a
0.14 ± 0.00a
0.38 ± 0.00a
PE
14.74 ± 0.04c
0.59 ± 0.02a
1.42 ± 0.01b
9.73 ± 0.10a
13.82 ± 0.01a
25.02 ± 0.31a
1.61 ± 0.05a
1.11 ± 0.01a
0.94 ± 0.01a
0.30 ± 0.00a
5.77 ± 0.17b
0.61 ± 0.17a
0.31 ± 0.00a
2.14 ± 0.00a
0.28 ± 0.00a
0.29 ± 0.01a
1.01 ± 0.06b
9.18 ± 0.21c
1.07 ± 0.17b
0.93 ± 0.14b
0.14 ± 0.03a
0.40 ± 0.03b
0.10 ± 0.03b
0.09 ± 0.03a
0.21 ± 0.02b
1.10 ± 0.03b
TAG
7.33 ± 0.15b
1.30 ± 0.02b
1.17 ± 0.02a
20.37 ± 0.53b
23.92 ± 0.16b
44.78 ± 0.65b
14.18 ± 0.55b
2.99 ± 0.00b
1.66 ± 0.01c
0.33 ± 0.01b
1.21 ± 0.03a
5.50 ± 0.38b
1.76 ± 0.01b
11.96 ± 0.49b
0.64 ± 0.02c
0.55 ± 0.01c
0.28 ± 0.01a
3.23 ± 0.02b
0.29 ± 0.03a
0.17 ± 0.02a
0.12 ± 0.02a
0.20 ± 0.01a
0.03 ± 0.01a
0.08 ± 0.01a
0.09 ± 0.00a
0.70 ± 0.04a
PC
B. sinensis
4.63 ± 0.10a
2.01 ± 0.03c
1.19 ± 0.02a
25.61 ± 0.26c
30.48 ± 0.11c
56.55 ± 0.31c
19.27 ± 0.06c
3.94 ± 0.17c
1.19 ± 0.04b
0.31 ± 0.00b
0.91 ± 0.02a
5.96 ± 0.03b
1.76 ± 0.04b
19.13 ± 0.25c
0.42 ± 0.01b
0.41 ± 0.00b
0.18 ± 0.03a
2.62 ± 0.09a
0.45 ± 0.16a
0.13 ± 0.05a
0.32 ± 0.11a
0.39 ± 0.20a
PE
12.75 ± 0.41c
0.88 ± 0.01a
1.29 ± 0.01c
13.59 ± 0.07a
17.54 ± 0.19b
31.86 ± 0.25a
4.14 ± 0.02a
1.92 ± 0.04a
0.11 ± 0.01b
2.50 ± 0.03b
0.38 ± 0.01c
4.55 ± 0.00b
2.11 ± 0.00a
1.64 ± 0.01ab
0.22 ± 0.00a
5.51 ± 0.11a
0.68 ± 0.01b
0.96 ± 0.00c
0.44 ± 0.00c
5.97 ± 0.09c
0.73 ± 0.01b
0.55 ± 0.01b
0.19 ± 0.02b
0.32 ± 0.06b
0.16 ± 0.02
0.09 ± 0.02a
0.07 ± 0.02
0.83 ± 0.03c
TAG
5.46 ± 0.13a
1.36 ± 0.02c
0.95 ± 0.08b
24.36 ± 1.31b
23.15 ± 0.64c
47.69 ± 0.67b
17.87 ± 1.41b
2.15 ± 0.04b
0.05 ± 0.04ab
3.52 ± 0.12c
0.10 ± 0.00a
0.66 ± 0.02a
4.88 ± 0.00c
1.37 ± 0.17a
0.24 ± 0.04b
14.11 ± 0.57c
0.45 ± 0.08a
0.24 ± 0.04a
0.12 ± 0.00b
1.69 ± 0.03a
0.17 ± 0.01a
0.10 ± 0.01a
0.07 ± 0.00a
0.13 ± 0.02a
0.13 ± 0.02a
0.67 ± 0.01b
PC
S. kneri Garman PE
7.88 ± 0.12b
1.29 ± 0.01b
0.51 ± 0.01a
31.84 ± 0.33c
16.38 ± 0.23a
48.21 ± 0.56c
26.79 ± 0.19c
2.90 ± 0.04c
0.03 ± 0.00a
1.31 ± 0.06a
0.19 ± 0.01b
0.64 ± 0.02a
3.47 ± 0.05b
1.97 ± 0.08b
0.36 ± 0.04b
6.04 ± 0.05b
0.32 ± 0.01a
0.37 ± 0.01b
0.09 ± 0.01a
3.78 ± 0.02b
0.38 ± 0.00a
Mean values ± SD (n = 3). Values in the same row with different letters for each species show significant differences (p < 0.05). TAG, triacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; total SFA, total saturated fatty acids; 17:1(a), 7-methyl-6-hexadecenoic acid; 17:1(b), 15-methyl-11-hexadecenoic acid; total cis MUFA, total cis monounsaturated fatty acids; total trans MUFA, total trans monounsaturated fatty acids; total trans DUFA, total trans diunsaturated fatty acids; total CLA, total conjugated linoleic acids; total PUFA, total polyunsaturated fatty acids; total n − 6 PUFA, total n − 6 polyunsaturated fatty acids; total n − 3 PUFA, total n − 3 polyunsaturated fatty acids; total OBCFA, total odd-branched chain fatty acids; , not detected.
a
0.03 ± 0.00a
0.11 ± 0.01b
tc18:2n-6
0.07 ± 0.00a
0.14 ± 0.05
0.11 ± 0.00b
0.32 ± 0.03a
0.46 ± 0.01b
PE
tt18:2n-6
0.72 ± 0.06c
total trans MUFA
PC
S. curriculus
ct18:2n-6
TAG
FA
Table 2. continued
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf204584t | J. Agric.Food Chem. 2012, 60, 1857−1864
Journal of Agricultural and Food Chemistry
Article
PE > PC > TAG. The ratio of (n − 6)/(n − 3) in all studied freshwater species ranged from 0.51 to 1.42; the ratio of ((n − 6) + (n − 3)) PUFA/SFA was found to be lower than 1 for TAG (0.48−0.88)) and higher than 1 for PL (1.26−2.03) in our samples. TAG. Among TAG of each selected fish, total SFA (31.59− 40.07%), and total MUFA (30.17−44.62%) predominated over total PUFA (18.38−31.86%) (Table 2). The major SFA was 16:0 from 16.29% to 21.10% followed by 18:0. Total cis MUFA varied from 29.34% in S. kneri Garman to 43.90% in S. curriculus, among which 18:1n-9 was the most abundant. The levels of 18:2n-6 and 18:3n-3 in B. sinensis were higher than any other fish, with 9.18% and 5.77%, respectively. E. ilishaeformis predominated in the content of 20:5n-3 (3.26%) and 22:6n-3 (6.01%) and S. kneri Garman of 20:4n-6 (5.51%) and 22:5n-3 (1.92%). We also observed a considerable amount of total odd-branched chain fatty acids (OBCFA) ranging from 5.94% for S. curriculus to 14.74% for B. sinensis. PL. On the contrary, the proportions of total PUFA had the maximum value (40.94−56.55%) and total MUFA (10.75− 22.80%) showed the minimum in PL fractions of each sample (Table 2). Total SFA exhibited modest fluctuation between 27.12% and 37.46% in both PC and PE of all fish, with PC containing relatively higher and lower amounts of 16:0 and 18:0 than the PE fraction, respectively (Table 2). Total cis MUFA in PL were lower than TAG with 18:1n-9 constituting the major isomer. Total PUFA was the richest fraction among PL from 40.94% to 47.81% for PC and from 48.21% to 56.55% for PE. 20:4n-6 and 22:6n-3 in PL (>10%) exhibited exclusively dominance over other PUFA of all species, with the exception of S. kneri Garman for 20:4n-6 (6.04%) in PE. S. curriculus predominated in the content of 20:5n-3 (PC, 7.54%; PE, 3.15%) and B. sinensis of 22:5n-3 (PC, 2.99%; PE, 3.94%). The total OBCFA in PL were consistently lower than TAG, with contents varying from 3.16% for S. curriculus in PC to 7.88% for S. kneri Garman in PE. FA Distributions in TAG and PL of Five Wild Freshwater Fish. Regarding positional FA distributions in TAG (Figure 1), 16:0, 22:5n-3, and 22:6n-3 were preferentially esterified in the sn-2-postion of all species while 18:0, 18:1n-9, 18:2n-6, 18:3n-3, 20:4n-6, and 20:5n-3 were mainly esterified in positions 1 and 3, with the exception of E. ilishaeformis and B. sinensis for 18:2n-6 and P. f ulvidraco for 20:4n-6 (Figure 1b−d). As indicated in Figure 2, stereospecific analysis of the TAG fraction showed preferential association of total SFA to the sn-2-position whereas total MUFA and PUFA to positions 1 and 3 in all samples . With respect to positional FA distributions in PC (Figure 3) and PE (Figure 4), 16:0, 18:0, 18:1n-9, and 18:3n-3 were preferentially esterified in the sn-1-position, followed in sequence by sn-2-position. 20:4n-6, 20:5n-3, 22:5n-3, and 22:6n-3 preferentially distributed in the sn-2-position of PL in all freshwater fish. Figure 2 shows that the levels of total PUFA were much higher in the sn-2-position (59.88−87.26%) than the sn-1-position (15.78−33.96%), whereas total SFA and total MUFA mainly occupied the sn-1-position (46.71−66.57% and 13.94−25.43%, respectively).
Figure 1. Positional distribution of major fatty acids in TAG of five wild freshwater fish from Poyang Lake (mol %, n = 3). Mean values with standard deviations plotted as bars: (a) S. curriculus; (b) E. ilishaeformis; (c) P. fulvidraco; (d) B. sinensis; (e) S. kneri Garman; TAG, triacylglycerol.
■
Figure 2. Positional distribution of total SFA, total MUFA, and total PUFA in TAG, PC, and PE fractions of five wild freshwater fish from Poyang Lake (mol %, n = 3). Mean values with standard deviations plotted as bars: (a) S. curriculus; (b) E. ilishaeformis; (c) P. fulvidraco; (d) B. sinensis; (e) S. kneri Garman; total SFA, total saturated fatty acids; total MUFA, total monounsaturated fatty acids; total PUFA, total polyunsaturated fatty acids; TAG, triacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine.
DISCUSSION PUFA. Table 2 shows that the total PUFA characterized the lowest and highest contents in TAG and PL among the fish selected, respectively, which were in good agreement with Simonetti et al., who reported the most notable PUFA were found in PL of cultured freshwater fish.22 It is interesting to 1861
dx.doi.org/10.1021/jf204584t | J. Agric.Food Chem. 2012, 60, 1857−1864
Journal of Agricultural and Food Chemistry
Article
(0.14−0.42%) from the Indus River6 and marine species (0.19−0.68%) from Malaysia.26 As a precursor for prostaglandin and thromboxane, 20:4n-6 could facilitate wound healing by influencing the process of the blood clot and attachment to the endothelial cells.27 Table 2 shows that the levels of 20:5n-3 and 22:6n-3 in TAG (3.26% and 6.01%, respectively), PC (6.12% and 15.44%, respectively), and PE (3.03% and 23.85%, respectively) from E. ilishaeformis were close to data (4.25− 11.6% for 20:5n-3; 6.15−16.1% for 22:6n-3) obtained from the Mauritanian coast.28 As reported in the Introduction, the variability of the fish fatty acid profile is due to location, environment, season, gender, diet, and species. Benedito-Palos et al.29 and Suzuki et al.30 proved that the high PUFA levels of fish depends on their diet rich in essential FA. A considerable amount of 18:2n-6 (17.26−27.88%), 18:3n-3 (5.51%), 20:5n-3 (2.03−2.95%), and 22:6n-3 (2.48−3.64%) were found in planktons of Poyang Lake (Lei et al., unpublished work). As a main water source of the Yangtze River and the largest freshwater lake in China, the unique location of Poyang Lake has a significant effect on its biodiversity. Hence, the high ratio of PUFA of wild freshwater fish in the study may be attributed to the distinctive environment of Poyang Lake. The physiological effects of n − 3 PUFA on the human body have been well documented;2,3 however, consumption (
